Project Summary Children, adolescents and young adults (CAYA) with a primary T-cell immunodeficiency (PID) and/or secondary T-cell immunodeficiency following allogeneic stem cell transplantation (AlloSCT) and a medically refractory CMV, ADV and/or EBV infection and/or who become intolerant to antiviral antibiotics have a dismal prognosis (?90% mortality rate). Refractory infections with CMV, ADV and EBV are uncommon and this represents an Orphan Disease (approximately 250 patients) in the United States. Strikingly, there is no FDA approved therapy for this rare patient population. Recently, adoptive T-cell therapy with viral specific cytotoxic T-lymphocytes (CTLs) is currently under early phase clinical investigations. This approach utilizes a rapid technique of viral specific CTL isolation following specific stimulation of donor peripheral blood mononuclear cells with viral-derived peptides followed by CTL isolation utilizing an INF-g Cytokine Capture System (Miltenyi Prodigy). In a small number of patients in single center studies, this approach has been feasible, safe and efficacious. The feasibility, safety and efficacy of this novel cellular therapy approach in this orphan population in larger multicenter prospective studies is presently lacking. We therefore hypothesize that partially HLA matched (haploidentical) related donor derived CMV, ADV and EBV CTLs manufactured utilizing the Miltenyi CliniMACS Prodigy Cytokine Capture System will be feasible, safe and effective in CAYA with either PID and/or SID following AlloSCT with medically refractory viral infections and/or those intolerant to antiviral antibiotics. We will investigate this hypothesis through three large multicenter viral CTL trials (IND17449) via the Viral CTL Consortium (VIRCTLC). The specific primary and secondary aims include (Brief): 1) feasibility and safety of CMV, ADV and EBV CTL production and infusion; 2) efficacy of viral specific CTLs (elimination of specific viral load by qRT-PCR below the lower limit of institutional values; 3) persistence of haploidentical donor viral CTL infusion; secondary: 4) probability of developing AGVHD; 5) probability of viral free and overall survival; 6) characterization of the genetic, proteomic and immunological properties of CMV, ADV and EBV CTLs; and 7) specific T-cell immune reconstitution, function and correlation to response to viral cell therapy. The experimental design including (brief): 1) viral CTL production by direct isolation following viral specific peptide stimulation and cytokine capture isolation; 2) three phase II open label prospective studies; 3) viral CTL validation and functionality; 4) viral CTL persistence by CD3 donor chimerism; and 5) viral CTL characterization by flow cytometry, mass cytometry by time of flight (CYTOF), single cell cytokine analysis, phosphoproteomics analysis, T-cell immunoprofiling, T cell receptor (TCR) diversity and frequency by Immunoseq, and single cell mRNA sequencing. The long term objectives are to obtain the feasibility, safety and efficacy of this therapy that will lead to FDA approval of CMV, ADV and EBV viral CTLs manufactured by the Miltenyi CliniMACS Prodigy Cytokine Capture System in this orphan patient population.